Proteinaceous emulsifier, process for preparing same and emulsion type cosmetic compositions containing same

ABSTRACT

A proteinaceous emulsifier can be obtained by reacting an amino acid ester with a hydrophilic protein in the presence of an endopeptidase to form decomposition products of the hydrophilic protein having a carboxyl end group joined to the amino group of the amino acid ester by amide linkage. 
     This proteinaceous emulsifier has a typical surfactant structure in which a hydrophobic portion consisting of the amino acid ester is attached to the rear end of a hydrophilic portion consisting of the protein, and is characterized by causing little irritation to the skin, exhibiting strong emulsifying power and possessing moisture retention properties. Thus, it is very suitable for use as an emulsifier in various cosmetics such as creams, milky lotions and the like.

This application is a continuation of U.S. Ser. No. 751,775, filed June25, 1985 now abandoned.

TECHNICAL FIELD

This invention relates to a proteinaceous emulsifier that causes littleirritation to the skin or the like, exhibits strong emulsifying powerand possesses moisture retention properties, a process for preparing thesame, as well as emulsion type cosmetic compositions. such as creams andthe like, containing the same.

BACKGROUND ART

Generally, emulsifiers are widely used in cosmetics, foods and the like.Emulsifiers used in cosmetics include anionic surfactants and nonionicsurfactants. However, these emulsifiers are disadvantageous in thatanionic surfactants are irritable to the skin and in that nonionicsurfactants fail to exhibit sufficient emulsifying power and are more orless irritable to the skin. Moreover, since these emulsifiers cannotimpart water retention properties to creams and the like, a moistureretentive agent such as glycerol or the like is added thereto accordingto the existing state of the art. However, this causes problems becausethe emulsion stability and/or feeling of the cosmetic may be altered.

Emulsifiers used in foods include sugar esters, fatty acidmonoglycerides and the like. These emulsifiers and disadvantageous inthat, since their emulsifying power is not so strong, they must be usedin large amounts to achieve a sufficient degree of emulsification.Accordingly, it is desired to provide an emulsifier that causes littleirritation to the skin, exhibits strong emulsifying power and possessesmoisture retention properties.

DISCLOSURE OF THE INVENTION

In view of the above-described state of the art, an object of thepresent invention is to provide an emulsifier which causes littleirritation to the skin, exhibits strong emulsifying power and possessesmoisture retention properties, a process for preparing such anemulsifier, and emulsion type cosmetic compositions containing such anemulsifier.

According to one feature of the present invention, there is provided aproteinaceous emulsifier composed of decomposition product of ahydrophilic protein having a carboxyl end group joined to the aminogroup of an amino acid ester by amide linkage.

According to another feature of the present invention, there is provideda process for preparing proteinacous emulsifiers which comprisesproviding an amino acid ester and a hydrophilic protein and reacting theamino acid ester with the hydrophilic protein in the presence of anendopeptidase to form a proteinaceous emulsifier composed ofdecomposition products of the hydrophilic protein having a carboxyl endgroup joined to the amino group of the amino acid ester.

According to still another feature of the present invention, there isprovided a process for preparing proteinaceous emulsifiers in which,where the aforesaid amino acid ester is represented by the generalformula (I) given below, the amino acid ester is reacted with thehydrophilic protein to form a decomposition product of the hydrophilicprotein having a carboxyl end group joined to the amino group of theamino acid ester, and the resulting reaction product is subjected todialysis.

According to a further feature of the present invention, there isprovided an emulsion type cosmetic composition containing, as theemulsifier, a proteinaceous emulsifier composed of a decompositionproduct of a hydrophilic protein having a carboxyl end group joined tothe amino group of an amino acid ester by amide linkage.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view illustrating a molecule of the proteinaceousemulsifier of the present invention;

FIGS. 2 and 3 are infrared absorption spectra of amino acid esterp-toluenesulfonates used in the preparation of emulsifiers in accordancewith the present invention;

FIGS. 4 and 5 are infrared absorption spectra of free amino acid estersused in the preparation of emulsifiers in accordance with the presentinvention;

FIGS. 6 and 8 are infrared absorption spectra of proteinaceousemulsifiers in accordance with the present invention;

FIGS. 9 to 11 are surface tension diagrams of proteinaceous emulsifiersin accordance with the present invention;

FIG. 12 is a diagram showing the relationship between emulsifierconcentration and absorbance (i.e., emulsifying power) for severalemulsifiers in accordance with the present invention which are composedof decomposition products of gelatin having the myristyl ester ofleucine, alanine or glycine joined thereto by amide linkage; and

FIG. 13 is an infrared absorption spectrum of a proteinaceous emulsifierin accordance with the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The proteinaceous emulsifier of the present invention is composed of adecomposition product of a hydrophilic protein having a carboxyl endgroup joined to the amino group of an amino acid ester by amide linkage.

Specific examples of the amino acid ester used in the present inventioninclude amino acid esters derived from alanine, glycine, tyrosine andtryptophan. They further include amino acid esters derived from aminoacids having a side chain at the a-position such as valine, leucine,isoleucine, etc.; basic amino acids such as lysine, arginine, histidine,etc.; and dibasic amino acids such as aspartic acid, glutamic acid, etc.Among the above-enumerated amino acid esters, esters of basic aminoacids are unstable and tend to become colored brown. Moreover, esters ofdibasic amino acids may form practically unseparable by-products duringthe so-called aminolysis reaction (or enzymatic reaction) in which anamino acid ester is joined, by amide linkage, to the carboxyl end groupof a decomposition product of a hydrophilic protein. Such by-productsare undesirable because they may give off an offensive odor and degradethe stability of the proteinaceous emulsifier. The alcohols constitutingthe above-enumerated amino acid esters include alcohols having 2 to 22carbon atoms, such as ethanol, propanol, butanol, heptanol, hexanol,octanol, decanol, dodecanol, myristyl alcohol, pentadecyl alcohol, cetylalcohol, heptadecyl alcohol, stearyl alcohol, arachyl alcohol, behenylalcohol and the like. They also include unsaturated alcohols such as2-pentenol-1, 11-dodecenol-1, oleyl alcohol and the like. In the case ofunsaturated alcohols, the trans isomer is preferred to the cis isomerbecause the former exhibits a higher degree of reactivity in theenzymatic reaction (or aminolysis reaction) involving the grafting of ahydrophilic protein. They further include branched-chain alcohols suchas isopropyl alcohol, isobutyl alcohol, tert-butyl alcohol and the like.In the case of branched-chain alcohols, the iso form of alcohols arepreferred because alcohols having an unduly bulky configuration exhibita low degree of reactivity in the enzymatic reaction (or aminolysisreaction).

In consideration of the above-described facts, preferred examples of theamino acid ester constituting the emulsifier of the present inventionare amino acid esters represented by the formula

    R.sub.1 CH(NH.sub.2)COOR.sub.2                             (1)

where R₁ is hydrogen, an alkyl group, an ω-hydroxyalkyl group, anaralkyl group or an ω-hydroxyaralkyl group and R₂ is a saturatedhydrocarbon or unsaturated hydrocarbon radical having 2 to 22 carbonatoms.

Among amino acid esters represented by the above formula (1), amino acidesters in which the substituent group R₁ has a high degree ofhydrophobicity are more preferred and these amino acid esters arerepresented by the formula

    R.sub.3 CH(NH.sub.2)COOR.sub.2                             (2)

where R₃ is a methyl, isopropyl, n-butyl, isobutyl, sec-butyl,1-hydroxyethyl, benzyl or p-hyroxybenzyl group and R₂ is as defined forformula (1).

Specific examples of amino acid esters represented by the above formula(2) include esters of alanine, valine, norleucine, leucine, isoleucine,threonine, phenylalanine and tyrosine.

Among amino acid esters represented by the above formula (2), it ispreferable to use an amino acid ester whose alcohol residue has 14 to 22carbon atoms. If an amino acid ester whose alcohol residue has less than14 carbon atoms is used, the resulting proteinaceous emulsifier willhave poor emulsifying power and unemulsified compositions containing itmay give off a slightly offensive odor after prolonged storage at hightemperature (e.g., at 45° C. for 6 months). On the other hand, if anamino acid ester whose alcohol residue has more than 22 carbon atoms isused, the amino acid ester will tend to have poor solubility and,therefore, exhibit a low degree of reactivity in the enzymatic reaction(or aminolysis reaction) with a hydrophilic protein. Accordingly, it ispreferable to use an amino acid ester whose alcohol residue has 14 to 22carbon atoms.

As is evident from Example 14 and Table 11 which will be given later,this tendency is marked especially where leucine is used as the aminoacid. Accordingly, where leucine is used as a constituent of theemulsifier of the present invention, it may safely be said that the mostpreferred leucine esters are ones represented by ##STR1## where R₅ is asaturated aliphatic hydrocarbon or unsaturated aliphatic hydrocarbonradical having 14 to 22 carbon atoms.

Furthermore, an investigation has been made as to the differences inemulsifying power based on the type of amino acid of the amino acidester constituting the emulsifier of the present invention. Thus, as isevident from Example 23 and 24 and FIG. 12 which will be given later,the most preferred amino acid esters are alanine esters represented by##STR2## where R₄ is a saturated aliphatic hydrocarbon or unsaturatedaliphatic hydrocarbon radical having 2 to 22 carbon atoms.

Second to such alanine esters are esters composed of leucine and analcohol having 14 to 22 carbon atoms, esters derived from isoleucine,and esters derived from tyrosine. Esters derived from glycine areslightly inferior in emulsifying power to the above-enumerated esters.

Although the amino acids constituting the above-enumerated estersinclude L-amino acids and D-amino acids, it is preferable to use anL-amino acid. If a D-amino acid is used, the yield of the resultingproteinaceous emulsifier will be reduced.

As the hydrophilic protein constituting the proteinaceous emulsifier ofthe present invention, gelatin is most preferred because of itshydrophilicity and safety, as well as its solubility in the reactionsystem, reaction efficiency and other characteristics desirable for theenzymatic reaction. In the second place, casein, sericin, solublecollagen and zein are preferable.

The above-described proteinaceous emulsifier can be prepared, forexample, in the following manner. Specifically, an amino acid is firstreacted with an alcohol to synthesize an amino acid ester to whichdecomposition products of a hydrophilic protein are to be joined.

In the synthesis of an amino acid ester, any of the well-knownesterification reactions may be properly chosen and effected by using astrong acid (such as sulfuric acid or the like) or a Lewis acid (such aszinc oxide, activated alumina, tetraisopropyl titanate or the like) asthe catalyst. However, more excellent results can be obtained byemploying the following process which proceeds by way ofp-toluenesulfonate of a free amino acid ester. That is, an amino acid isreacted with an alcohol in the presence of the p-toluenesulfonate, aBronsted acid catalyst, to form the p-toluenesulfonate of an amino acidester.

Since the esterification reaction is carried out in the presence ofp-toluenesulfonic acid, the amino acid ester is not obtained in the freestate, but in the form of an amino acid ester p-toluenesulfonate. In thepractice of the present invention, this salt can also be used in thereaction with decomposition products of a hydrophilic protein and,therefore, can be a raw material for the preparation of the emulsifierof the present invention.

However, when reacted with a hydrophilic protein in the presence of anendopeptidase, this amino acid ester p-toluenesulfonate is poorlysoluble in the solvent comprising a mixture of a buffer solution andacetone. Especially where the alcohol residue constituting the aminoacid ester has 14 or more carbon atoms, it becomes more difficult toestablish a homogeneous system as the chain is lengthened, and the yieldof the resulting proteinaceous emulsifier is less than desirable.Moreover, the amino acid ester p-toluenesulfonate can hardly be freed ofimpurities such as unreacted amino acid, p-toluenesulfonic acid and thelike, even though the product resulting from the esterification reactionis purified by recrystallizing it repeatedly from suitable solvents suchas acetone and the like to remove unreacted impurities. Accordingly, ifthe amino acid ester p-toluenesulfonate is used as such, the degree ofconversion into the desired proteinaceous emulsifier will be low, andwhat is more, the resulting proteinaceous emulsifier will becontaminated with appreciable amounts of impurities. Thus, in order toobtain a high pure proteinaceous emulsifier, it will be necessary towash the product repeatedly with hot acetone or the like in the finalstep of the process. For these reasons, it is advantageous to subjectthe amino acid ester p-toluenesulfonate to an alkali treatment (undersalting-out conditions, if desired) and use the resulting free aminoacid ester in the reaction with a hydrophilic protein. As the alkalinemedium, an aqueous solution of NaOH or KOH is preferably used. Thealkali concentration is preferably in the range of 0.01N to 5N and morepreferably in the range of 0.05N to 2N. It is also preferable to carryout the aforesaid alkali treatment under salting-out conditions, i.e.,in the coexistence of a salt. The salts useful for salting-out purposesinclude alkali metal salts, ammonium salts and the like. Specificexamples thereof include sodium chloride, potassium chloride, potassiumcarbonate, potassium phosphate, ammonium sulfate, sodium sulfate and thelike. However, sodium chloride and potassium chloride are preferred. Thesalt concentration used for salting-out purposes is usually in the rangeof 0.5 to 25% and preferably in the range of 1 to 10%. While theaforesaid amino acid ester p-toluenesulfonate is a solid, the free aminoacid ester thus obtained is in the form of an oil or a low-meltingsolid. Thus, during the succeeding reaction with a hydrophilic protein,the free amino acid ester is very easily soluble in the reaction solventcomprising, for example, a mixture of a buffer solution and acetone.Accordingly, the reaction can be carried out in a homogeneous system.

However, where an alanine ester represented by the above formula (3) isused as the amino acid ester, desirable results can be obtained by usingthe resulting alanine ester p-toluenesulfonate as such, i.e., withouteliminating p-toluenesulfonic acid from the alanine esterp-toluenesulfonate. Specifically, alanine esters, though in the form ofp-toluenesulfonates, exhibit excellent solubility. Thus, even if analanine ester p-toluenesulfonate is used as such, any residues (such asunreacted alanine, p-toluenesulfonic acid and the like) remaining in thereaction system are effectively removed, as contrasted with the case inwhich other amino acid esters are used. Accordingly, the desired productcan be obtained in a highly purified state.

It is to be understood that, in order to distinguish between free aminoacid esters and their p-toluenesulfonates in the following descriptiongiven in this specification, the term "free amino acid ester"exclusively denotes compounds represented by the above general formula(1), while the term "amino acid ester" is used in its broadest sense andcomprehends both such compounds and their p-toluenesulfonates.

The amino acid ester obtained in the above-described manner is thenreacted with a hydrophilic protein as described below in the presence ofan endopeptidase as described below.

(Hydrophilic Proteins)

Casein, gelatin, sericin, soluble collagen, zein, serum albumin,lactalbumin and egg albumin may be used alone or in combination. Amongthese proteins, other proteins that sericin are commercially availablereagents. Sericin can be obtained by extracting cut cocoons with hotwater and freeze-drying the extract. It is most preferable to usegelatin because of its hydrophilicity, safety and other desirableproperties, as well as its solubility in the reaction system, reactionefficiency and other characteristics desirable for the enzymaticreaction.

(Endopeptidases)

Endopeptidases have the effect of splitting protein at centrally locatedpoints of its molecule, and include serine proteases, thiol proteases,carboxyl proteases, metal proteases and other endopeptidases. Amongthem, thiol proteases are most preferably used. Useful thiol proteasesinclude papain, ficin, cathepsin B, kiwi fruit protease, bromelain,chymopapain, cathepsin L, yeast proteinase B, cathepsin S andTZ-peptidase, which may be used alone or in combination.

When the amino acid ester is reacted with a hydrophilic protein in thepresence of an endopeptidase, the hydrophilic protein is split atcentrally located points by the action of the endopeptidase and, at thesame time, the carboxyl end group of the resulting decompositionproducts thereof is joined to the amino group of the amino acid ester byamide linkage. Thus, there is obtained a proteinaceous emulsifier inaccordance with the present invention.

If a free amino acid ester is used in the above-described reaction witha hydrophilic protein, the free amino acid ester can be uniformlydispersed in the solvent of the reaction system because of its desirableform (i.e., an oil or a low-melting solid). Accordingly, when a freeamino acid ester is reacted with a hydrophilic protein in the presenceof an endopeptidase, the reaction efficiency is so markedly improvedthat a highly pure proteinaceous emulsifier having excellentemulsification characteristics can be obtained in high yield.

One suitable method for after-treatment of the resulting enzymaticreaction product is dialysis. Specifically, after the endopeptidaseremaining in the enzymatic reaction product is deactivated under acidconditions, the enzymetic reaction product is subjected to dialysis. Thedialysis may be carried out by use of a hollow-fiber ultrafiltrationmembrane, especially contained in a cylindrical housing, to obtaindesirable results. As the material of the hollow-fiber ultrafiltrationmembrane, there may be used cellulose acetate, cellulose,polyacrylonitrile, ethylene vinyl alcohol, polyvinyl alcohol, Cuprofanmembrane (manufactured by Enka GmbH, West Germany) and the like.Preferably, cellulose, polyacrylonitrile or Cuprofan membrane is chosen.

In dialyzing and concentrating the enzymetic reaction product by use ofsuch a hollow-fiber ultrafiltration membrane, the hollow-fiberultrafiltration membrane is preferably kept at a temperature of 25° C.to 50° C. and more preferably at a temperature of 35° C. to 45° C.

By using such a hollow-fiber ultrafiltration device for purposes ofdialysis and concentration, the enzymatic reaction product can bedeodorized perfectly and, moreover, can be desalted and concentrated ina very short period of time.

Furthermore, since the water content has been reduced in the aforesaidconcentration step, a subsequent drying step can also be carried out inan economical manner and in a short period of time. Thus, an odorlessproteinaceous emulsifier can be obtained in a short period of time andin an economical manner, and further in high yield.

A schematic view illustrating a molecule of the emulsifier of thepresent invention is shown in FIG. 1. In this figure, reference numeral1 designates a portion consisting of a decomposition product of ahydrophilic protein and reference numeral 2 designates a portionconsisting of an amino acid ester which includes an amino acid moiety 3and an alcohol residue moiety 4. In the proteinaceous emulsifierrepresented by the aforesaid schematic view, the portion 1 consisting ofa decomposition product of a hydrophilic protein constitutes ahydrophilic portion and the portion 2 consisting of an amino acid esterconstitutes a hydrophobic protein.

Since the hydrophilic portion 1 consists of a protein inherently havingstrong hydrophilicity and the hydrophobic portion 2 consists of ahydrophobic amino acid ester, this proteinaceous emulsifier has atypical surfactant structure (in which the front end of the hydrophobicportion 2 is attached to the rear end of the hydrophilic portion 1 andno extra hydrophobic portions are attached to intermediate points of thehydrophilic portion 1) and, therefore, exhibits strong emulsifyingpower. Moreover, since its emulsifying power is scarcely influenced bytemperature due to the polymeric structure of the protein portion, thisemulsifier does not show a reduction in emulsifying power, especially atlow temperatures. Furthermore, since the aforesaid hydrophilic portion 1has a relatively large molecular weight (of about 500 or more, andusually several thousands), the whole emulsifier also has a largemolecular weight, thus causing little irritation to the skin or thelike. In addition, this emulsifier possesses satisfactory moistureretention properties because the hydrophilic portion 1 consists of aprotein inherently having moisture-retaining power. Accordingly, if thisemulsifier is used in cosmetics, especially in creams, satisfactorywater retention properties can be imparted thereto without using anyconventional moisture-retaining agents such as polyhydric alcohols andthe like.

Where the emulsifier of the present invention is prepared by using afree amino acid ester represented by formula (1) as the amino acidester, the efficiency of the reaction between the free amino acid esterand the hydrophilic protein is significantly enhanced, so that theresulting proteinaceous emulsifier has markedly improved emulsificationcharacteristics and hence strong emulsifying power.

Furthermore, where the enzymatic reaction product is subjected todialysis as an after-treatment, the resulting proteinaceous emulsifieris odorless. Accordingly, even in absence of perfume, it can be usedwithout giving any disagreeable feeling. Alternatively, it can beperfurmed without spoiling the inherent odor of the perfume.

Thus, since the above-described proteinaceous emulsifier causes littleirritation to the skin or the like, exhibits strong emulsifying powerand possesses moisture retention properties, desirable effects areproduced when it is used, for example, in cosmetics. When it is utilizedas an emulsifer in foods, its strong emulsifying power makes it possibleto reduce the amount of emulsifier used. Moreover, since most of theconstituents of this emulsifier are derived from natural materials, noproblem will arise even if it is ingested repeatedly.

Especially when the proteinaceous emulsifier is prepared by using analiphatic alcohol ester of alanine represented by formula (3) as a rawmaterial, it has excellent emulsifying power. Moreover, it has apleasant odor as contrasted with proteinaceous emulsifiers derived fromother amino acids. Furthermore, in the preparation thereof, such analiphatic alcohol ester of alanine exhibits good solubility and otherexcellent properties. Thus the efficiency of the reaction between theester and the hydrophilic protein is greatly enhanced to result in amarked improvement in yield.

As described above, the proteinaceous emulsifiers of the presentinvention have a typical surfactant structure in which a hydrophobicportion consisting of an amino acid ester is attached to the rear end ofa hydrophilic portion consisting of a protein and are characterized bycausing very little irritation to the skin, strong emulsifying power andmoisture retention properties. Thus, when they are used in cosmetics,especially in creams and the like, water retention properties can beimparted thereto without using any polyhydric alcohol such as glycerolor the like, and the occurrence of skin diseases in persons having anirritable skin can be decreased markedly. Moreover, when they areutilized in foods, their strong emulsifying power makes it possible toreduce the amount of emulsifier used. Furthermore, according to theprocess of the present invention, an amino acid ester is reacted with ahydrophilic protein in the presence of an endopeptidase and, as soon asthe hydrophilic protein is split, the amino acid ester is joined to thecut end of its decomposition product. Thus, an emulsifier having atypical surfactant structure in which a hydrophobic portion is attachedto the rear end of a hydrophilic portion can be obtained with ease andin very high yield.

The emulsion type cosmetic compositions of the present invention containa proteinaceous emulsifier as described above. The methods by which theemulsion type cosmetic compositions are made and the proteinaceousemulsifier is incorporated thereinto are not critical, and any ofvarious conventional methods may be employed. Although various creamsare typical of the emulsion type cosmetic compositions, it is to beunderstood that they include all types of cosmetics (such as milkylotions and the like) subjected to an emulsification procedure using anemulsifier.

In the emulsion type cosmetic compositions of the present invention, aproteinaceous emulsifier as described above may be used alone or incombination with any of conventional emulsifiers as described at thebeginning of this specification.

As described above, the emulsion type cosmetic compositions of thepresent invention contain a proteinaceous emulsifier that has a typicalsurfactant structure in which a hydrophobic portion consisting of anamino acid ester is attached to the rear end of a hydrophilic portionconsisting of a protein and is characterized by causing very littleirritation to the skin, strong emulsifying power and moisture retentionproperties. More specifically, because of the moisture-retaining effectof the aforesaid emulsifier, these cosmetic compositions havesatisfactory water retention properties without using any polyhydricalcohol such as glycerol or the like. Moreover, since the aforesaidemulsifier causes very little irritation to the skin, these cosmeticcompositions give rise to no skin diseases even if a person havingsensitive skin uses them continually. Furthermore, the strongemulsifying power of the aforesaid emulsifier makes it possible toreduce the amount of emulsifier used in the cosmetic compositions, whichalso contributes to the decrease in irritation to the skin.

The present invention is further illustrated by the following examples.

EXAMPLE 1 [PROTEINACEOUS EMULSIFIER 1]

An amino acid ester was prepared in the manner described below and thenused in an enzymatic reaction.

(Synthesis of an Amino Acid Ester P-Toluenesulfonate)

0.05 mole of leucine was taken as an amino acid, 0.055 mole ofp-toluenesulfonic acid hydrate was taken, and 0.075 mole of oleylalcohol was taken as an alcohol. After 10 ml of benzene was addedthereto as a solvent, these materials were stirred and mixed well. Theresulting reaction mixture was heated at the reflux temperature ofbenzene to carry out the esterification reaction. In this case, waterwas formed with the progress of the esterification reaction and releasedin the form of an azeotropic mixture with benzene, which wascontinuously removed from the reaction system. Thus, the reaction wascompleted in about 5-10 hours. Then, the reaction mixture wasconcentrated under reduced pressure to remove the benzene used assolvent. After the addition of ether and petroleum ether, theprecipitated crystals were collected by filtration. These crude crystalswere recrystallized from acetone, ether, petroleum ether and the like.An infrared absorption spectrum of the leucine oleyl esterp-toluenesulfonate thus obtained is shown in FIG. 2. In this figure, thepeak 5 indicates the absorption of light by a para-substituted phenylgroup, the peaks 6 and 7 by the ester linkage, the peak 8 by sulfonicacid, and the peaks 9 and 10 by a primary amine salt.

(Enzymatic Reaction)

82 g of gelatin as a hydrophilic protein was dissolved in 200 g of a 1Mcarbonate buffer solution (pH 9.0). After the addition of 50 g ofacetone, the resulting mixture was warmed in a water bath at 35° C. andhomogenized by adequate stirring. Then, 30 g (i.e., 1 mole per 1,000 gof the gelatin) of leucine oleyl ester p-toluenesulfonate obtained inthe above-described manner was added thereto and homogenized by adequatestirring. In this case, it is desirable from the viewpoint of reactionefficiency to use the amino acid ester (i.e., leucine oleyl ester) in anamount of 1 mole per 1,000 g of the hydrophilic protein (i.e., gelatin).Thereafter, 20 mg of 2-mercaptoethanol was added thereto, followed by 40mg of crystalline papain (manufactured by Sigma Co.) as anendopeptidase. The resulting reaction mixture was stirred for 60minutes, after which the reaction was stopped by bringing its pH to 2with 1N hydrochloric acid. The reaction mixture was placed in acellophane tube (or dialysis tube), dialyzed against running water fortwo days and nights, and then freeze-dried. The product was washed withhot acetone to remove any unreacted leucine oleyl ester and therebyobtain the desired proteinaceous emulsifier (hereinafter referred to asProteinaceous Emulsifier 1). An infrared absorption spectrum ofProteinaceous Emulsifier 1 thus obtained is shown in FIG. 6. In thisfigure, the peak 11 indicate the absorption of light by protein and thepeak 12 by hydrogen.

EXAMPLE 2 [PROTEINACEOUS EMULSIFIER2] (Synthesis of an Amino Acid EsterP-Toluenesulfonate)

Glycine myrityl ester p-toluenesulfonate was prepared in substantiallythe same manner as in Example 1, except that glycine was used in placeof leucine and myristyl alcohol was used in place of oleyl alcohol.

(Enzymatic Reaction)

Instead of 82 g of gelatin, 30 g of sericin was dissolved in 200 g of a1M carbonate buffer solution. After the addition of 50 g of acetone, theresulting mixture was warmed on a water bath at 35° C. and homogenizedby stirring. Then, 8.6 g (i.e., 1 mole per 1,000 g of the sericin) ofglycine myristyl ester p-toluenesulfonate was added thereto andhomogenized by stirring. Thereafter, an enzymatic reaction was carriedout in the same manner as in Example 1 except that the amount ofcrystalline papain used was decreased to 20 mg. Thus, there was obtainedProteinaceous Emulsifier 2.

Then, using Tween 80 as a control, the surface tension characteristicsof Proteinaceous Emulsifier 1 obtained in the above-described Example 1are shown in FIG. 9. In this figure, the concentration-surface tensioncurve of Proteinaceous Emulsifier 1 obtained in Example 1 is denoted byA and that of the control is denoted by B. By comparison of the curves Aand B, it can be seen that Proteinaceous Emulsifier 1 obtained inExample 1 is markedly superior to Tween 80 in surface activity.

Then, the performance of the proteinaceous emulsifiers obtained in theabove-described manner was evaluated by using them in a cosmeticcomposition and a food. In these cases, it was preferable from theviewpoint of effectiveness to use them in an amount of 1 to 2% byweight.

EXAMPLE 3 [SKIN CREAM]

Using the formulation given in Table 1 below, a skin cream was madeaccording to conventional procedure. Specifically, Solution 1 wasprepared by mixing the components 1, 2 and 3 given in Table 1 below at80° C. until the mixture became homogeneous. On the other hand, Solution2 was prepared by mixing the undermentioned components 4 to 6 at 80° C.until the mixture became homogeneous. Thereafter, while Solution 2 wasbeing stirred in a homomixer, Solution 2 was added thereto andemulsified therein. The resulting emulsion was cooled, during which theundermentioned component 7 was added thereto at 70° C. When thetemperature fell to 30° C., the stirring was discontinued to obtain askin cream. The skin cream thus obtained was an O/W emulsion. On theother hand, another skin cream (Comparative Example 1) was made by usingthe undermentioned emulsifier (given in Comparative Example 1 ofTable 1) in place of Proteinaceous Emulsifier 1.

                  TABLE 1                                                         ______________________________________                                        (parts by weight)                                                             ______________________________________                                                                 Comparative                                          Component     Example 3  Example 1                                            ______________________________________                                        1.   Cetyl alcohol                                                                              10         10                                               2.   Octyldodecyl 1.5        1.5                                                   myristate                                                                3.   Liquid paraffin                                                                            20.0       20.0                                             4.   Emulsifier   Proteinaceous                                                                            Polyoxyethylene                                                    Emulsifier 1                                                                             cetyl ether (10E.0)                                                of Example 1                                                                             2                                                                  2.0        Glycerol mono-                                                                stearate                                                                      1                                                5.   Methylparaben                                                                              0.3        0.3                                              6.   Purifier water                                                                             66.0       65.0                                             7.   Perfume      0.2        0.2                                              ______________________________________                                    

The results of performance tests of the resulting skin creams are shownin Table 2. As can be seen from Table 2, the skin cream of Example 3 wasmarkedly superior to that of Comparative Example 1 in feeling,organoleptic effects and physiological effects on the skin.

                  TABLE 2                                                         ______________________________________                                                                     Comparative                                                      Example 3    Example 1                                        Skin Cream      (persons/    (persons/                                        Test item       persons)     persons)                                         ______________________________________                                        (1) Appearance                                                                    Immediately after                                                                             White, creamy                                                                              Same as                                          preparation                  left                                             After storage at 45° C.                                                                "            Same as                                          for 6 months                 left                                             After storage at 5° C.                                                                 "            Same as                                          for 6 months                 left                                         (2) Smell                                                                         Immediately after                                                                             Good         Same as                                          preparation                  left                                             After storage at 45° C.                                                                "            Same as                                          for 6 months                 left                                             After storage at 5° C.                                                                 "            Same as                                          for 6 months                 left                                         (3) Feeling                                                                       Stickiness      0/20         7/20                                             Agglomeration   0/20         5/20                                         (4) Organoleptic effects                                                          Finer texture   16/20        8/20                                             Better complexion                                                                             15/20        7/20                                             Tightened skin  18/20        9/20                                         (5) Physiological effects                                                         on skin                                                                       Improvement of dry                                                            skin                                                                          Effective       16/20        8/20                                             Slightly effective                                                                            3/20         4/20                                             Ineffective     1/20         8/20                                             Moisture-retaining                                                            effect                                                                        Effective       15/20        8/20                                             Slightly effective                                                                            3/20         5/20                                             Ineffective     2/20         7/20                                             Amelioration of                                                               keratinization                                                                Effective       17/20        7/20                                             Slightly effective                                                                            2/20         4/20                                             Ineffective     1/20         9/20                                         ______________________________________                                    

The results shown in Table 2 above were obtained according to thefollowing test procedures.

(1) Appearance

The cream of Example 3 and the cream of Comparative Example 1 werestored at 45° C. or 5° C. for 6 months. Their appearance before andafter storage was comparatively evaluated by 5 expert examiners.

(2) Smell

The cream of Example 3 and the cream of Comparative Example 1 was storedat 45° C. or 5° C. for 6 months. Their smell before and after storagewas comparatively evaluated by 5 expert examiners.

(3) Feeling

Twenty female panelists were instructed to apply about 0.5 g of each ofthe two samples to the respective sides of the face. The occurrence of"stickiness" and/or "agglomeration" immediately after application wasevaluated by the panelists themselves.

(4) Organoleptic effects

Twenty female panelists were instructed to apply about 0.5 g each of thetwo samples to the respective sides of the face. This application wasmade once a day and continued for 2 weeks. The state of the skin beforeand after testing was evaluated by the panelists themselves.

(5) Evaluation of the improvement of dry skin

Using 20 subjects of middle or advanced age having a dry skin on thelegs, the effect of the creams was examined by applying them continuallyfor 2 weeks. Specifically, about 1 g of a sample to be tested wasapplied once a day to a test area of the left leg of each subject. Thestate of the skin before and after testing was rated according to thefollowing criteria. The right leg, to which no sample was applied wasused as control.

Criteria for Rating the Dryness of the Skin

-: Normal.

±: Slightly dry, no desquamation.

+: Dry, slight desquamation.

++: Dry, moderate desquamation.

+++: Dry, marked desquamation.

The ratings of the test area before and after testing and the ratings ofthe control area were compared. The sample was judged to be "effective"when the dryness of the skin was improved by two or more steps (e.g.,+→-or ++→±); "slightly effective" when the dryness was improved by onestep; and "ineffective" when the dryness remained unchanged. No subjectsshowed an aggravation of the dryness of the skin.

(6) Evaluation of the moisture-retaining effect

Skin conductance was measured with a skin impedance meter (manufacturedby IBS Co.). The moisture retension effect was evaluated by a short-termtest in which the measurement was made 12 hours after a singleapplication of a sample and by a continual-use test in which themeasurement was made after a sample was applied once a day for 2 weeks.The tests were regarded as negative when the increase in skinconductance was less than 50%. The sample was judged to be "effective"when both the short-term test and the continual-use test were positive;"slightly effective" when only one of the tests was positive; and"ineffective" when both of the tests were negative.

(7) Evaluation of the amelioration of keratinization (or the improvementof resistance of scaling-off of horny layer)

After the above-described test for evaluating the improvement of dryskin, a piece of Scotch tape (Nichiban mending tape) was applied to theskin of the test area and then peeled off. The state of any horney layeradhering to the tape was closely examined with a scanning electronmicroscope. Thus, the amelioration of keratinization was evaluated byscoring the resistance of the skin to scaling-off of the horny layeraccording to the following criteria.

Criteria for Evaluating the Amelioration of Keratinization (or theImprovement of Resistance to Scaling-Off of Horny Layers)

Score 1: No scales are observed.

Score 2: Small-sized scales are scattered.

Score 3: Small- to medium-sized scales are abundant.

Score 4: Large-sized scales are abundant.

The score of the test area examined after continual application for 2weeks was compared with that of the control area. The sample was judgedto be "effective" when the difference between the two scores was 2 ormore; "slightly effective" when the difference was 1; and "ineffective"when the difference was 0.

There were no cases in which the score of the test area was greater thanthat of the control area.

APPLICATION EXAMPLE 1 [ICE CREAM]

An ice cream was made by mixing raw materials in the proportion shownbelow and treating the mixture according to conventional procedure.

    ______________________________________                                        Formulation                                                                   ______________________________________                                        Granulated sugar     15     parts by weight                                   Milk powder          10     parts by weight                                   Hydrogenated coconut oil                                                                           5      parts by weight                                   Millet jelly powder  5      parts by weight                                   (manufactured by Meiji Nyugyo                                                 K. K.)                                                                        CMC                  0.2    parts by weight                                   Proteinaceous Emulsifier 2                                                                         0.3    parts by weight                                   (obtained in Example 2)                                                       Water                64.5   parts by weight                                   ______________________________________                                    

On the other hand, another ice cream (Comparative Example 2) was made byusing the same amount of monostearin in place of ProteinaceousEmulsifier 2.

Then, the ice creams thus obtained were comparatively evaluated. As aresult, when compared with the ice cream of Application Example 1, theice cream of Comparative Example 2 had a poor gloss immediately afterthe manufacture thereof, and this poor gloss still persisted at the timeof shipment and imparted thereto a quite unattractive appearance. Incontrast, the ice cream of Application Example 1 had a good glossimmediately after the manufacture thereof and this good gloss lasted atleast till the time of shipment. Moreover, the ice cream of ComparativeExample 2 had the disadvantage that a sufficient overrun could not beachieved in the over-running step of the manufacturing process. However,such a disadvantage was not encountered in the ice cream of ApplicationExample 1.

EXAMPLE 4 [PROTEINACEOUS EMULSIFIER 3]

Proteinaceous Emulsifier 3 was prepared in the same manner as describedfor Proteinaceous Emulsifier 2, except that lauryl alcohol was used inplace of myristyl alcohol.

EXAMPLE 5 [PROTEINACEOUS EMULSIFIER 4]

Proteinaceous Emulsifier 4 was prepared in the same manner as describedfor Proteinaceous Emulsifier 2, except that octyl alcohol was used inplace of myristyl alcohol.

Then, emulsion type cosmetic compositions were made using ProteinaceousEmulsifiers 1-4 prepared in the above-described manner. For thispurpose, it has been found desirable from the viewpoint of effectivenessto use all of them in an amount of 1 to 3% by weight.

EXAMPLE 6 AND COMPARATIVE EXAMPLE 3 [MASSAGE CREAM]

In the formulations given in Table 3 below, Solution 1 was prepared bymixing the oily components 1 and 2 at 80° C. until the mixture becamehomogeneous. On the other hand, Solution 2 was prepared by mixing thewater-soluble components 3, 4 and 5 at 80° C. until the mixture becamehomogeneous. Thereafter, while the aforesaid Solution 2 was beingstirred in a homomixer, Solution 2 was added thereto and emulsifiedtherein. The resulting emulsion was cooled, during which thebelow-mentioned component 6 was added thereto at 70° C. When thetemperature fell to 30° C., the stirring was discontinued. Thus, therewere obtained two massage creams of the O/W emulsion type.

                  TABLE 3                                                         ______________________________________                                        (parts by weight)                                                             ______________________________________                                                                  Comparative                                         Component      Example 6  Example 3                                           ______________________________________                                        1.   Liquid paraffin                                                                             50         50                                              2.   Cetyl alcohol 5          5                                               3.   Emulsifier    Proteinaceous                                                                            Polyoxyethylene                                                    Emulsifier 1                                                                             sorbitan mono-                                                     3          oleate (20E.0)                                                                2.5                                                                           Sorbitan mono-                                                                oleate                                                                        2.5                                             4.   Methylparaben 0.2        0.2                                             5.   Purifier water                                                                              41.5       39.5                                            6.   Perfume       0.3        0.3                                             ______________________________________                                    

EXAMPLE 7 AND COMPARATIVE EXAMPLE 4 [SKIN MILK]

Using the formulations given in Table 4 below, two skin milks were madein the same manner as described in Example 6 except that Solution 1 wasprepared from the oily components 1, 2 and 3 and Solution 2 was preparedfrom the water-soluble components 4, 5, 6 and 7.

                  TABLE 4                                                         ______________________________________                                        (parts by weight)                                                             ______________________________________                                                                  Comparative                                         Component      Example 7  Example 4                                           ______________________________________                                        1.   Cetyl alcohol 6           6                                              2.   Castor oil    2           2                                              3.   Liquid paraffin                                                                             20          20                                             4.   Emulsifier    Proteinaceous                                                                             Polyoxyethylene                                                   emulsifier 1                                                                              cetyl ether                                                       1.5         (5.5E.0)                                                                      2                                                                             Sorbitan                                                                      sesquioleate                                                                  1                                              5.   Propylene glycol                                                                            5           5                                              6.   Methylparaben 0.5         0.2                                            7.   Purifier water                                                                              65.1        63.5                                           8.   Perfume       0.2         0.2                                            ______________________________________                                    

EXAMPLE 8 AND COMPARATIVE EXAMPLE 5 [SKIN MILK]

Using the formulations given in Table 5 below, two skin milks were madein the same manner as described in Example 6 except that Solution 1 wasprepared from the oily components 1, 2 and 3 and Solution 2 was preparedfrom the water-soluble components 4, 5 and 6 .

                  TABLE 5                                                         ______________________________________                                        (parts by weight)                                                             ______________________________________                                                                  Comparative                                         Component      Example 8  Example 5                                           ______________________________________                                        1.   Cetyl alcohol 5          5                                               2.   Octyldodecyl  1.5        1.5                                                  myristate                                                                3.   Liquid paraffin                                                                             20.0       20.0                                            4.   Emulsifier    Proteinaceous                                                                            Polyoxyethylene                                                    Emulsifier 1                                                                             cetyl ether                                                        2.0        (10E.0)                                                                       2                                                                             Glycerol mono-                                                                stearate                                                                      1                                               5.   Methylparaben 0.3        0.3                                             6.   Purifier water                                                                              71.0       70.0                                            7.   Perfume       0.2        0.2                                             ______________________________________                                    

EXAMPLE 9 AND COMPARATIVE EXAMPLE 6 [CLEANSING CREAM]

Using the formulations given in Table 6 below, two cleansing creams weremade in the same manner as described in Example 6 except that Solution 1was prepared from the oily components 1 and 2 and Solution 2 wasprepared from the water-soluble components 3, 4 and 5.

                  TABLE 6                                                         ______________________________________                                        (parts by weight)                                                             ______________________________________                                                                  Comparative                                         Component      Example 9  Example 6                                           ______________________________________                                        1.   Cetyl alcohol 5          5                                               2.   Liquid paraffin                                                                             60         60                                              3.   Emulsifier    Proteinaceous                                                                            Polyoxyethylene                                                    Emulsifier 2                                                                             monostearate                                                       3          (2.0E.0)                                                                      3                                                                             Sorbitan                                                                      sesquioleate                                                                  2.5                                             4.   Methylparaben 0.2        0.2                                             5.   Purifier water                                                                              31.5       29.0                                            6.   Perfume       0.3        0.3                                             ______________________________________                                    

EXAMPLE 10 AND COMPARATIVE EXAMPLE 7 [CLEANSING MILK]

Using the formulations given in Table 7 below, two cleansing milks weremade in the same manner as described in Example 6 except that Solution 1was prepared from the oily components 1, 2 and 3 and Solution 2 wasprepared from the water-soluble components 4, 5 and 6.

                  TABLE 7                                                         ______________________________________                                        (parts by weight)                                                             ______________________________________                                                                  Comparative                                         Component      Example 10 Example 7                                           ______________________________________                                        1.   Liquid paraffin                                                                             50         50                                              2.   Isocetyl      2          2                                                    myristate                                                                3.   Xanthane gum  1          1                                               4.   Emulsifier    Proteinaceous                                                                            Sorbitan                                                           Emulsifier 2                                                                             sesquioleate                                                       1          5                                                                             Polyoxyethylene                                                               monostearate                                                                  (20E.0)                                                                       1.5                                             5.   Methylparaben 0.2        0.2                                             6.   Purifier water                                                                              45.5       43.5                                            7.   Perfume       0.3        0.3                                             ______________________________________                                    

EXAMPLE 11 AND COMPARATIVE EXAMPLE 8 [FOUNDATION CREAM]

Using the formulations given in Table 8 below, two foundation creamswere made in the same manner as described in Example 6 except thatSolution 1 was prepared from the oily components 1 and 2 and Solution 2was prepared from the water-soluble components 3 to 9.

                  TABLE 8                                                         ______________________________________                                        (parts by weight)                                                             ______________________________________                                                                  Comparative                                         Component      Example 11 Example 8                                           ______________________________________                                        1.   Liquid paraffin                                                                             45         45                                              2.   Ceresin       5          5                                               3.   Emulsifier    Proteinaceous                                                                            Polyoxyethylene                                                    Emulsifier 2                                                                             cetyl ether                                                        1.4        (5.5E.0)                                                                      2                                                                             Sorbitan                                                                      sequioleate                                                                   1                                               4.   Methylparaben 0.3        0.3                                             5.   Titanium oxide                                                                              2.5        2.5                                             6.   Kaolin        1.25       1.25                                            7.   Talc          1.1        1.1                                             8.   Yellow iron   0.4        0.4                                                  oxide                                                                    9.   Purified water                                                                              43.05      41.45                                           ______________________________________                                    

EXAMPLE 12 [CLEANSING CREAM]

A cleansing cream was made in the same manner as described in Example 9except that Proteinaceous Emulsifier 3 was used in place ofProteinaceous Emulsifier 2.

EXAMPLE 13 [CLEANSING CREAM]

A cleansing cream was made in the same manner as described in Example 9except that Proteinaceous Emulsifier 4 was used in place ofProteinaceous Emulsifier 2.

The results of performance tests of the various creams obtained in theabove-described examples and comparative examples are shown in Table 9.As can be seen from Table 9, the creams obtained in the examples weremarkedly superior to those obtained in the comparative examples, infeeling, organoleptic effects and physiological effects on the skin.

These tests were carried out according to the same procedures asdescribed in Example 3.

    TABLE 9       Compar-  Compar-  Compar-  Compar-  Compar-  Compar-    ative  ative     ative  ative  ative  ative Example Example Example Example Example     Example Example Example Example Example Example Example Example Example     6 3 7 4 8 5 9* 6* 10* 7* 11* 8* 12* 13*        (1) Appearance                Immediately after preparation White,     Same as White, Same as Same as Same as White Same as White, Same as     Skin- Same as White, Same as   creamy left milky left left left creamy     left milky left colored, left creamy left             creamy  After     storage at 45° C. for White, Same as White, Same as Same as Same     as White, Slight White, Same as Skin- Same as White, Same as  6 months     creamy left milky left left left creamy separa- milky left colored, left     creamy left          tion   creamy (2)  Immediately after preparaion     Good Good Good Good Good Good Good Good Good Good Good Good Good Good     After storage at 45° C. for " " " " " " " " " " " " Slightly Same     as  6 months             offensive left (3) Feeling  Stickiness (pers./pe     rs.) 0/20 7/20 0/20 6/20 0/20 7/20 0/20 6/20 0/20 5/20 0/20 7/20 0/20     0/20  Agglomeration (pers./pers.) 0/20 5/20 0/20 6/20 0/20 0/20 0/20     5/20 0/20 0/20 0/20 0/20 0/20 0/20 (4) Organoleptic effects  Finer     texture (pers./pers.) 16/20  8/20 16/20  5/20 17/20  7/20 16/20  6/20     17/20  5/20 16/20  7/20 16/20 16/20  Better complexion 15/20 7/20 15/20     5/20 16/20  6/20 17/20  5/20 16/20  6/20 15/20  6/20 15/20 16/20     (pers./pers.)  Tightened skin (pers./pers.) 18/20  9/20 18/20  7/20     17/20  8/20 17/20  8/20 17/20  7/20 18/20       7/20 17/20 17/20 (5) Physiological effects on skin  Improvement of dry     skin  Effective (pers./pers.) 17/20  8/20 16/20  7/20 15/20  17/20 8/20     16/20  7/20 15/20 7/20  Slightly effective 2/20 4/20 3/20 5/20 3/20 2/20     4/20 3/20 5/20 3/20 6/20  (pers./pers.)  Ineffective (pers./pers.) 1/20     8/20 1/20 8/20 2/20 1/20 8/20 1/20 8/20 2/20 7/20  Moisture-retaining     effect  Effective (pers./pers.) 16/20  8/20 15/20  7/20 16/20  16/20     8/20 15/20  7/20 16/20 5/20  Slightly effective 3/20 5/20 3/20 3/20 3/20     3/20 5/20 3/20 3/20 3/20 6/20  (pers./pers.)  Ineffective (pers./pers.)     1/20 7/20 2/20 10/20  1/20 1/20 7/20 2/20 10/20  1/20 9/20  Amelioration     of keratinization  Effective (pers./pers.) 17/20  7/20 18/20  6/20 15/20      17/20  7/20 18/20  6/20 15/20  6/20  Slightly effective 2/20 4/20 1/20     6/20 3/20 2/20 4/20 1/20 6/20 3/20 4/20 (pers./pers.)  Ineffective     (pers./pers.) 1/20 9/20 1/20 8/20 2/20 1/20 9/20 1/20 8/20 2/20 10/20     *Examples 9-13 and Comparative Examples 6-8 are concerned with cleansing     creams, cleansing milks and foundation creams, which are intended to be     washed away after application or have the effect of fixing other cosmetic     to the face. Thus, their physiological effects on the skin were not teste     because such effects are no more than secondary.

EXAMPLE 14 [PROTEINACEOUS EMULSIFIER 5]

A free amino acid ester was prepared in the manner described below andthen used in an enzymatic reaction.

(Synthesis of Free Amino Acid Ester

0.05 mole of leucine was used as an amino acid, 0.055 mole ofp-toluenesulfonic acid hydrate was used, and 0.075 mole of myristylalcohol was used as an alcohol. After 200 ml of benzene was addedthereto as a solvent, these materials were stirred and mixed well. Theresulting reaction mixture was heated at the reflux temperature ofbenzene to carry out the esterification reaction. In this case, waterwas formed with the progress of the esterification reaction and releasedin the form of an azeotropic mixture with benzene, which wascontinuously removed from the reaction system. Thus, the reaction wascompleted in about 5-10 hours. Then, the reaction mixture wasconcentrated under reduced pressure to remove the benzene used assolvent. After the addition of ether and petroleum ether, theprecipitated crystals were collected by filtration. These crude crystalswere recrystallized from acetone, ether, petroleum ether and the like.

The resulting leucine myristyl ester p-toluenesulfonate was dissolvedagain in 200 ml of benzene, and this solution was treated three timeswith 300--ml portions of 0.1N NaOH. Thereafter, the benzene layer wasisolated and washed thoroughly with water. After washing with water, thebenzene layer was isolated and dried overnight over anhydrous sodiumsulfate. After drying, the benzene layer was filtered and the resultingbenzene solution was evaporated to remove the solvent. Thus, purifiedfree leucine myristyl ester was obtained in the form of an oilymaterial.

An infrared absorption spectrum of the leucine oleyl ester thus obtainedis shown in FIG. 4. In this figure, the peaks 5 and 6 indicate the C═Ostretching and C--O stretching vibrations originating from the esterlinkage. The peaks 7, 8 and 9 indicate the NH stretching, NH deformationand C--N stretching vibrations, respectively, of a primary amine. Thepeak 10 indicates the CH deformation vibration.

(Enzymatic Reaction)

50 g of gelatin as a hydrophilic protein was dissolved in 200 ml of a 1Mcarbonate buffer solution (pH 9.0). After the addition of 50 g ofacetone, the resulting mixture was warmed on a water bath at 37° C. andhomogenized by adequate stirring. Then, 16.4 g (i.e., 1 mole per 1,000 gof the gelatin) of leucine myristyl ester obtained in theabove-described manner was added thereto and homogenized by adequatestirring. In this case, it is desirable from the viewpoint of reactionefficiency to use the amino acid ester (i.e., leucine myristyl ester) inan amount of 1 mole per 1,000 g of the hydrophilic protein (i.e.,gelatin). Thereafter, 438 mg of L-cysteine hydrochloride (manufacturedby Wako Pure Chemicals Co., Ltd.) was added thereto, followed by 25 mgof papain (manufactured by Sigma Co.) as an endopeptidase. The resultingreaction mixture was stirred for 15 minutes, after which the reactionwas stopped by bringing its pH to 2 with 2N hydrochloric acid. Thereaction mixture was placed in a cellophane tube (or dialysis tube),dialyzed against running water for two days and nights, and thenfreeze-dried. The product was washed with hot acetone to remove anyunreacted leucine myristyl ester and thereby obtain the desiredproteinaceous emulsifier (hereinafter referred to as ProteinaceousEmulsifier 5). An infrared absorption spectrum of ProteinaceousEmulsifier 5 thus obtained is shown in FIG. 7. In this figure, the peak11 indicates the absorption of light by protein and the peak 12 and 13by hydrogen.

Other proteinaceous emulsifiers were prepared in the same manner as inthe above example except that the p-toluenesulfonates of esters composedof leucine and each of the alcohols shown in Table 10 below and suchesters in the free state were used in place of leucine myristyl esterp-toluenesulfonate and free leucine myristyl ester, respectively.

With regard to the cases in which the alcohol residue is lauryl,myristyl, cetyl, stearyl, arachyl and behenyl, the yield of theproteinaceous emulsifier obtained by reacting an amino acid esterp-toluenesulfonate with a hydrophilic protein and the yield of theproteinaceous emulsifier obtained by reacting a free amino acid esterwith a hydrophilic protein were comparatively examined. The results thusobtained are shown in Table 10. It can be seen from this table that, atany chain length of the alcohol residue, the yield of the proteinaceousemulsifier obtained by reacting a free amino acid ester with ahydrophilic protein is significantly higher.

                                      TABLE 10                                    __________________________________________________________________________                       Alcohol residue                                                               Lauryl                                                                            Myristyl                                                                           Cetyl                                                                             Stearyl                                                                           Arachyl                                                                            Behenyl                              Ester              (C.sub.12)                                                                        (C.sub.14)                                                                         (C.sub.16)                                                                        (C.sub.18)                                                                        (C.sub.20)                                                                         (C.sub.22)                           __________________________________________________________________________    Leucine ester                                                                        Amount used*.sup.1 (g)                                                                    9.43                                                                              9.99 10.55                                                                             11.12                                                                             11.68                                                                              12.24                                p-toluene-                                                                           Unreacted residue (g)                                                                     1.15                                                                              2.68 4.15                                                                              6.22                                                                              6.74 6.98                                 sulfonate                                                                            Yield*.sup.2 (mole %)                                                                     87.8                                                                              73.7 60.7                                                                              44.1                                                                              42.3 43.0                                 Free leucine                                                                         Amount used (g)                                                                           5.99                                                                              6.55 7.1 7.67                                                                              8.23 8.79                                 ester  Unreacted residue*.sup.3 (g)                                                              0.55                                                                              1.3  2.01                                                                              3.13                                                                              3.73 4.02                                        Yield*.sup.2 (mole %)                                                                     89.5                                                                              82.15                                                                              74.4                                                                              62.8                                                                              58.4 57.8                                 __________________________________________________________________________     *.sup.1 The amount of reactant used per 20 g of the hydrophilic protein.      *.sup.2 The yield (mole %) of the resulting proteinaceous emulsifier.         *.sup.3 This residue was obtained in the form of a leucine ester              hydrochloride.                                                           

The yields shown in Table 10 above were calculated as follows: In thefinal step, each proteinaceous emulsifier was washed three times withhot acetone. The acetone solutions obtained by filtration were combinedand evaporated to remove the solvent. Then, the yield (mole %) wascalculated from the amount of the unreacted leucine esterp-toluenesulfonate or leucine ester hydrochloride thus obtained. (Thesame shall apply hereinafter.) ##EQU1##

The following tests for emulsifying power were carried out on four ofthe proteinaceous emulsifiers shown in Table 10 above, i.e., theproteinaceous emulsifiers obtained by reacting free leucine laurylester, free leucine myristyl ester, free leucine cetyl ester or freeleucine stearyl ester with gelatin in the above example.

(Tests for Emulsifying Power)

25.0 parts by weight of liquid paraffin, 1.0 part by weight of aproteinaceous emulsifier and 74.0 parts by weight of water were mixed.The resulting mixture was homogenized at 9,000 rpm at 80° C. for 2minutes and then cooled to room temperature. The emulsion so formed wascentrifuged at 500 rpm for 25 minutes and the volume of the resultingcream layer was measured. The emulsifying power of the proteinaceousemulsifier was expressed as the volume of the cream layer divided by thetotal volume.

The results thus obtained are shown in Table 11 below.

                  TABLE 11                                                        ______________________________________                                        Alcohol residue of free                                                       leucine ester constituting                                                    proteinaceous emulsifier                                                                       Emulsifying power                                            ______________________________________                                        Lauryl (C.sub.12)                                                                             0.55                                                          Myristyl (C.sub.14)                                                                           0.80                                                          Cetyl (C.sub.16)                                                                              0.75                                                          Stearyl (C.sub.18)                                                                            0.65                                                          ______________________________________                                    

As can be seen from the above table, the proteinaceous emulsifiersderived from leucine esters composed of leucine and an alcohol having 14or more carbon atoms are much superior in emulsifying power to those inwhich the alcohol has 12 or less carbon atoms.

EXAMPLE 15 [PROTEINACEOUS EMULSIFIER 6]

A free amino acid ester was prepared in the manner described below andthen used in an enzymatic reaction.

(Synthesis of a Free Amino Acid Ester)

0.05 mole of glycine was used as an amino acid, 0.055 mole ofp-toluenesulfonic acid hydrate was used, and 0.075 mole of myristylalcohol was used as an alcohol. After 200 ml of benzene was addedthereto as a solvent, these materials were stirred and mixed well. Theresulting reaction mixture was heated at the reflux temperature ofbenzene to carry out the esterification reaction. In this case, waterwas formed with the progress of the esterification reaction and releasedin the form of an azeotropic mixture with benzene, which wascontinuously removed from the reaction system. Thus, the reaction wascompleted in about 7 hours. Then, the reaction mixture was concentratedunder reduced pressure to remove the benzene used as solvent. After theaddition of ether and petroleum ether, the precipitated crystals werecollected by filtration. These crude crystals were recrystallized fromacetone, ether, petroleum ether and the like. The resulting glycinestearyl ester p-toluenesulfonate was dissolved again in 200 ml ofbenzene, and this solution was subjected to an alkali treatment undersalting-out conditions by the addition of 300 ml of a 3% NaCl solutioncontaining 0.1N NaOH. This alkali treatment was repeated three times.Thereafter, the benzene layer was isolated and washed thoroughly with a3% NaCl solution. Thereafter, the benzene layer was treated in the samemanner as in Example 14 to obtain purified free glycine stearyl ester.

(Enzymatic Reaction)

Instead of 50 g of gelatin, 30 g of zein was dissolved in 200 ml of a 1Mcarbonate buffer solution. After the addition of 50 ml of acetone, thereslting mixture was warmed on a water bath at 35° C. and homogenized bystirring. Then, 10.7 g (i.e., 1 mole per 1,000 g of the zein) of glycinestearyl ester which had previously been melted on a water bath at 50° C.was added thereto and homogenized by stirring. Thereafter, an enzymaticreaction was carried out in the same manner as in Example 14 except thatthe amount of crystalline papain used was decreased to 20 mg. Thus,there was obtained Proteinaceous Emulsifier 6.

Then, using gelatin as a control, the surface tension characteristics ofProteinaceous Emulsifier 6 obtained in the above-described Example 14are shown in FIG. 10. In this figure, the concentration-surface tensioncurve of Proteinaceous Emulsifier 6 obtained in Example 14 is denoted byA and that of the control is denoted by B. By comparison of the curves Aand B, it can be seen that Proteinaceous Emulsifier 6 obtained inExample 14 is markedly superior to gelatin in surface activity.

EXAMPLE 6 [PROTEINACEOUS EMULSIFIER 7]

A free amino acid ester was prepared in the manner described below andthen used in an enzymatic reaction.

(Synthesis of an Amino Acid Ester)

0.05 mole of leucine was taken as an amino acid, 0.055 mole ofp-toluenesulfonic acid hydrate was taken, and 0.075 mole of myristylalcohol was taken as an alcohol. After benzene was added thereto as asolvent, these materials were stirred and mixed well. The resultingreaction mixture was heated at the reflux temperature of benzene tocarry out the esterification reaction. In this case, water was formedwith the progress of the esterification reaction and released in theform of an azeotropic mixture with benzene, which was continuouslyremoved from the reaction system. Thus, the reaction was completed inabout 5-10 hours.

After being cooled to room temperature, the reaction mixture was washedthree times with a 3% NaCl solution containing 0.3N NaOH. Thereafter,the benzene layer was isolated and washed thoroughly with a 3% NaClsolution until it became neutral. After being lightly washed with water,the benzene layer was isolated and dried overnight over anhydrous sodiumsulfate. After drying, the benzene layer was filtered and the resultingbenzene solution was evaporated to remove the solvent. Thus, purifiedfree leucine myristyl ester was obtained in the form of an oilymaterial.

(Enzymatic Reaction)

120 g of gelatin as a hydrophilic protein was dissolved in 480 ml of a1M carbonate buffer solution (pH 9.0). After the addition of 120 ml ofacetone, the resulting mixture was warmed on a water bath at 37° C. andhomogenized by adequate stirring. Then, 39.3 g (i.e., 1 mole per 1,000 gof the gelatin) of leucine myristyl ester obtained in theabove-described manner was added thereto and homogenized by stirring. Inthis case, it is desirable from the viewpoint of reaction efficiency touse the amino acid ester (i.e., leucine myristyl ester) in an amount of1 mole per 1,000 g of the hydrophilic protein (i.e., gelatin).Thereafter, 1.05 g of L-cysteine hydrochloride (manufactured by WakoPure Chemicals Co., Ltd.) was added thereto, followed by 60 mg of papain(manufactured by Sigma Co.) as an endopeptidase. The resulting reactionmixture was stirred for 15 minutes, after which the reaction was stoppedby bringing its pH to 2 with 2N hydrochloric acid. The volume of thereaction mixture was about 1.5 liters. While being kept at about 40° C.,this reaction mixture was fed to a hollow-fiber ultrafiltration device(using a Cuprofan mambrane and having an internal fiber diameter of200μ, a membrane thickness of 11μ and an effective membrane area of 1.5m²) at a flow rate of about 10 ml/min. As the outer fluid, warm water atabout 40° C. was fed at a flow rate of about 1 liter/min. Aftercompletion of the dialysis, the internal pressure of the samehollow-fiber ultrafiltration device was adjusted to about 2 kg/cm² andthe dialyzed solution was concentrated under the same conditions asdescribed above. Finally, about 700 ml of a concentrated solution wasobtained.

This concentrated solution was dried with a spray dryer to obtain about8.5 g of an odorless proteinaceous emulsifier (hereinafter referred toas Proteinaceous Emulsifier 7).

EXAMPLE 17 [PROTEINACEOUS EMULSIFIER 8]

A free amino acid ester was prepared in the manner described below andthen used in an enzymatic reaction.

(Synthesis of a Free Amino Acid Ester)

0.05 mole of glycine was used as an amino acid, 0.055 mole ofp-toluenesulfonic acid hydrate was used, and 0.075 mole of stearylalcohol was used as an alcohol. After benzene was added thereto as asolvent, these materials were stirred and mixed well. The resultingreaction mixture was heated at the reflux temperature of benzene tocarry out the esterification reaction. In this case, water was formedwith the progress of the esterification reaction and released in theform of an azeotropic mixture with benzene, which is continuouslyremoved from the reaction system. Thus, the reaction was completed inabout 7 hours. After being cooled to room temperature, the reactionmixture was subjected to an alkali treatment under salting-outconditions by the addition of 300 ml of a 3% NaCl solution containing0.5N NaOH. This alkali treatment was repeated three times. Thereafter,the benzene layer was isolated and washed thoroughly with a 3% NaClsolution. Thereafter, the benzene layer was treated in the same manneras in Example 16 to obtain purified free glycine stearyl ester in theform of an oily material.

(Enzymatic Reaction)

Instead of 120 g of gelatin, 70 g of zein was dissolved in 480 ml of a1M carbonate buffer solution. After the addition of 120 ml of acetone,the resulting mixture was warmed on a water bath at 35° C. andhomogenized by stirring. Then, 19.0 g (i.e., 1 mole per 1,000 g of thezein) of glycine stearyl ester which had previously been melted on awater bath at 50° C. was added thereto and homogenized by stirring.Thereafter, 1.05 g of L-cysteine hydrochloride (manufactured by WakoPure Chemicals Co., Ltd.) was added thereto, followed by 45 mg of papain(manufactured by Sigma Co.) as an endopeptidase. The resulting reactionmixture was stirred for 15 minutes, after which the reaction was stoppedby bringing its pH to 2 with 2N hydrochloric acid. The volume of thereaction mixture was about 1.5 liters. While being kept at about 40° C.,this reaction mixture was fed to a hollow-fiber ultrafiltration device(using a cellulose membrane and having an internal fiber diameter of200μ, a membrane thickness of 11μ and an effective membrane area of 2.4m²) at a flow rate of about 10 ml/min. As the outer fluid, warm water atabout 40° C. was fed at a flow rate of about 1 liter/min. Aftercompletion of the dialysis, the internal pressure of the samehollow-fiber ultrafiltration device was adjusted to about 2 kg/cm² andthe dialyzed solution was concentrated under the same conditions asdescribed above. Finally, about 600 ml of a concentration solution wasobtained.

This concentrated solution was dried with a spray dryer to obtain about63 g of an odorless proteinaceous emulsifier (hereinafter referred to asProteinaceous Emulsifier 8).

In Examples 16 and 17, the efficiency of the dialysis process using ahollow-fiber ultrafiltration device was comparatively evaluated bymeasuring the conductivity of the reaction mixture with a desk typedigital conductivity meter (Model AO-6; manufactured by ElectrochemicalInstrument Co., Ltd.). The results thus obtained are shown in Table 12.

                  TABLE 12                                                        ______________________________________                                                            Conductivity                                              ______________________________________                                        Undialyzed reaction                                                                           Example 16                                                                              4.3 mΩ                                        mixture         Example 17                                                                              4.8 mΩ                                        Dialyzed reaction                                                                             Example 16                                                                              0.22 mΩ                                       mixture         Example 17                                                                              0.25 mΩ                                       Tap water             0.19 mΩ                                           ______________________________________                                    

As can be seen from Table 12, the conductivity of the reaction mixturehaving undergone a single dialysis is almost equal to that of tap waterused as the outer fluid, indicating that a satisfactory dialysisefficiency was achieved.

As demonstrated above, when the reaction product is deodorized by adialysis and concentration process using a hollow-fiber ultrafiltrationmembrane, a proteinaceous emulsifier characterized by causing littleirritation to the skin or the like exhibiting strong emulsifying powerand possessing moisture retention properties can be obtained in aperfectly deodorized state. Moreover, this can be accomplished in ashort period of time and in an economical manner, and further, in highyield.

EXAMPLE 18 [PROTEINACEOUS EMULSIFIER 9]

An ester of alanine was prepared in the manner described below and thenused in an enzymatic reaction.

(Synthesis of an L-alanine ester p-toluenesulfonate)

4.46 g (0.05 mole) of L-alanine was used, 11.4 g (0.06 mole) ofp-toluenesulfonic acid hydrate was used, and 11.79 g (0.055 mole) ofmyristyl alcohol was used as an alcohol. After 200 ml of benzene wasadded thereto as a solvent, these materials were stirred and mixed well.The resulting mixture was heated at the reflux temperature of benzene tocarry out the esterification reaction. In this case, water was formedwith the progress of the esterification reaction and released in theform of an azeotropic mixture with benzene, which was continuouslyremoved from the reaction system. Thus, the reaction was completed inabout 7-15 hours. Then, the reaction mixture was concentrated underreduced pressure to remove the benzene used as solvent. After theaddition of ether and petroleum ether, the precipitated crystals werecollected by filtration. These crude crystals were recrystallized fromacetone, ether, petroleum ether and the like to obtain 21.74 g (95%yield) of L-alanine myristyl ester p-toluenesulfonate. An infraredabsorption spectrum of the L-alanine myristyl ester p-toluenesulfonatethus obtained is shown in FIG. 3. In this figure, the peak 5 indicatesthe absorption of light by a parasubstituted phenyl group, the peaks 6and 7 by the ester linkage, the peak 8 by sulfonic acid, the peaks 9 and10 by a primary amine salt, and the peak 11 by the C═O stretchingvibration originating from the ester linkage.

(Enzymatic Reaction)

80 g of gelatin as a hydrophilic protein was dissolved in 200 g of a 1Mcarbonate buffer solution (pH 9.0). After the addition of 50 g ofacetone, the resulting mixture was warmed on a water bath at 37° C. andhomogenized by adequate stirring. Then, 37 g (i.e., 1 mole per 1,000 gof the gelatin) of L-alanine myristyl ester p-toluenesulfonate obtainedin the above-described manner was added thereto and homogenized byadequate stirring. In this case, it is desirable from the viewpoint ofreaction efficiency to use the amino acid ester (i.e., L-alaninemyristyl ester) in an amount of 1 mole per 1,000 g of the hydrophilicprotein (i.e., gelatin). Thereafter, 20 mg of 2-mercaptoethanol wasadded, followed by 40 mg of crystalline papain (manufactured by SigmaCo.) as an endopeptidase were added thereto. The resulting reactionmixture was stirred for 15 minutes, after which the reaction was stoppedby bringing its pH to 2 with 2N hydrochloric acid. The reaction mixturewas placed in a cellophane tube (or dialysis tubes), dialyzed againstrunning water for two days and nights, and then freeze-dried. Theproduct is washed with hot acetone to remove any unreacted L-alaninemyristyl ester and thereby obtain 83 g of the desired proteinaceousemulsifier (hereinafter referred to as Proteinaceous Emulsifier 9).

EXAMPLE 19 [PROTEINACEOUS EMULSIFIER 10]

A free L-alanine ester was prepared in the manner described below andthen used in an enzymatic reaction.

(Synthesis of a Free L-Alanine Ester)

L-Alanine cetyl ester p-toluenesulfonate was synthesized in the samemanner as described in Example 18 for the synthesis of an L-alaninealkyl ester p-toluenesulfonate, except that 13.3 g (0.055 mole) of cetylalcohol was used in place of myristyl alcohol. The resulting L-alaninecetyl ester p-toluenesulfonate was dissolved again in 200 ml of benzene,and this solution was treated three times with 300-ml portions of 0.1NNaOH. Thereafter, the benzene layer was isolated and washed thoroughlywith water. After washing with water, the benzene layer was isolated anddried overnight over anhydrous sodium sulfate. After drying, the benzenelayer was filtered and the resulting benzene solution was evaporated toremove the solvent. Thus, there was obtained 14.86 g (94.8% yield) ofpurified free L-alanine cetyl ester in the form of an oily material. Aninfrared absorption spectrum of the free L-alanine cetyl ester thusobtained is shown in FIG. 5. In this figure, the peaks 12 and 13indicate the C═O stretching and C--O stretching vibrations originatingfrom the ester linkage. The peaks 14, 15 and 16 indicate the NHstretching, NH deformation and C--N stretching vibrations, respectively,of a primary amine. The peak 17 indicates the CH deformation vibration.

(Enzymatic Reaction)

50 g of gelatin as a hydrophilic protein was dissolved in 200 ml of a 1Mcarbonate buffer solution (pH 9.0). After the addition of 50 ml ofacetone, the resulting mixture was warmed on a water bath at 37° C. andhomogenized by adequate stirring. Then, 15.7 g (i.e., 1 mole per 1,000 gof the gelatin) of L-alanine cetyl ester obtained in the above-describedmanner was added thereto and homogenized by adequate stirring. In thiscase, it is desirable from the viewpoint of reaction efficiency to usethe amino acid ester (i.e., L-alanine cetyl ester) in an amount of 1mole per 1,000 g of the hydrophilic protein (i.e., gelatin). Thereafter,438 mg of L-cysteine hydrochloride (manufactured by Wako Pure ChemicalsCo., Ltd.) was added thereto, followed by 25 mg of papain (manufacturedby Sigma Co.) as an endopeptidase. The resulting reaction mixture wasstirred for 15 minutes, after which the reaction was stopped by bringingits pH to 2 with 2N hydrochloric acid. The reaction mixture was placedin a cellophane tube (or dialysis tube), dialyzed against running waterfor two days and nights, and then freeze-dried. The product was washedwith hot acetone to remove any unreacted leucine myristyl ester andthereby obtain 48.9 g of the desired proteinaceous emulsifier(hereinafter referred to as Proteinaceous Emulsifier 10). An infraredabsorption spectrum of Proteinaceous Emulsifier 5 thus obtained is shownin FIG. 8. In this figure, the peak 18 indicates the absorption of lightby protein and the peak 19 by hydrogen.

EXAMPLE 20 [(PROTEINACEOUS EMULSIFIER 11]

Proteinaceous Emulsifier 11 was prepared in the same manner as inExample 19 except that a free L-alanine ester was synthesized accordingto the following procedure.

(Synthesis of a Free L-Alanine Ester)

4.46 g (0.05 mole) of L-alanine was taken, and 13.3 g (0.055 mole) ofcetyl alcohol was taken as an alcohol. After 200 ml of benzene was addedthereto as a solvent, these materials were stirred and mixed. After theaddition of 4.9 g (0.05 mole) of sulfuric acid as an acid catalyst, theresulting reaction mixture was heated under reflux. In this case, waterwas formed with the progress of the esterification reaction and releasedin the form of an azeotropic mixture with benzene, which wascontinuously removed from the reaction system. Thus, the reaction wascompleted in about 7 hours (at an internal temperature of 80.5° C.).After being cooled to room temperature, the reaction mixture was pouredinto a separating funnel and treated with 300 ml of a mixture of 0.2NNaOH and 6% NaCl solutions. This treatment was repeated three times.Thereafter, the benzene layer was isolated and washed thoroughly withwater. After washing with water, the benzene layer was isolated anddried overnight over anhydrous sodium sulfate. Thereafter, the benzenelayer was filtered and the resulting benzene solution was evaporated toremove the solvent. Thus, there was obtained 13.5 g (86.1% yield) ofpurified L-alanine cetyl ester in the form of an oily material.

EXAMPLE 21 [PROTEINACEOUS EMULSIFIER 12]

A free L-alanine ester was prepared in the manner described below andthen used in an enzymatic reaction.

(Synthesis of a Free L-Alanine Ester)

L-Alanine stearyl ester p-toluenesulfonate was synthesized in the samemanner as described in Example 18 for the synthesis of an L-alanineester p-toluenesulfonate, except that 11.4 g (0.06 mole) of stearylalcohol was used in place of myristyl alcohol. The resulting L-alaninestearyl ester p-toluenesulfonate was dissolved again in 200 ml ofbenzene, and this solution was treated three times with 300-ml portionsof 0.4N NaOH. Thereafter, the benzene layer was isolated and washedthoroughly with water. After washing with water, the benzene layer wasisolated and dried overnight over anhydrous sodium sulfate. Afterdrying, the benzene layer was filtered and the resulting benzenesolution was evaporated to remove the solvent. Thus, there was obtained16.15 g (94.4% yield) of purified free L-alanine stearyl ester in theform of an oily material.

(Enzymatic Reaction)

120 ml of acetone was added to 480 ml of purified water and theresulting mixture was kept at 37° C. with stirring. 120 g of gelatin asa hydrophilic protein was added thereto. Then, 41 g (i.e., 1 mole per1,000 g of the gelatin) of L-alanine stearyl ester obtained in theabove-described manner was added thereto and homogenized by adequatestirring. The resulting reaction mixture was adjusted to pH 9.0 with 1NNa₂ CO₃. On the other hand, an enzyme solution was prepared by adding1.05 g of L-cysteine hydrochloride monohydrate and 62.5 mg of papain(manufactured by Sigma Co.) to 5 ml of purified water and preincubatingthis mixture at 37° C. for an hour with stirring. After the addition ofthis enzyme solution, the aforesaid reaction mixture was stirred at 37°C. for 20 minutes. Then, the reaction was stopped by bringing the pH to2.0 with 2N hydrochloric acid. The total volume of the reaction mixturewas about 1.5 liters. While being kept at about 40° C., this reactionmixture was fed to a hollow-fiber ultrafiltration device (using aCuprofan membrane and having an internal fiber diameter of 200μ, amembrane thickness of 11μ and an effective membrane area of 1.5 m²) at aflow rate of about 10 ml/min. As the outer fluid, warm water at about40° C. was fed at a flow rate of about 1 liter/min. After completion ofthe dialysis, the internal pressure of the same hollow-fiberultrafiltration device was adjusted to about 2 kg/cm² and the dialyzedsolution was concentrated under the same conditions as described above.Finally, about 800 ml of a concentrated solution was obtained. Thisconcentrated solution was dried with a spray drier to obtain about 123 gof an odorless proteinaceous emulsifier (hereinafter referred to asProteinaceous Emulsifier 12).

EXAMPLE 22 [PROTEINACEOUS EMULSIFIER 13]

Proteinaceous Emulsifier 13 was prepared by using L-leucine in place ofL-alanine. Specifically, L-leucine myristyl ester p-toluenesulfonate wassynthesized in the same manner as described in Example 18 for thesynthesis of an L-alanine ester p-toluenesulfonate, except thatL-leucine was used in place of L-alanine. The resultingp-toluenesulfonate was dissolved in 200 ml of benzene, and this solutionwas treated three times with 300-ml portions of 0.1N NaOH. Thereafter,the benzene layer was isolated and washed thoroughly with water. Afterwashing with water, the benzene layer was isolated and dried overnightover anhydrous sodium sulfate. After drying, the benzene layer wasfiltered and the resulting benzene solution was evaporated to remove thesolvent. Thus, purified L-leucine myristyl ester was obtained in theform of an oily material. Using this ester, an enzymatic reaction wascarried out in the same manner as in Example 18 to obtain ProteinaceousEmulsifier 13.

Then, using gelatin as a control, the surface tension characteristics(measured at 30° C.) of the proteinaceous emulsifiers obtained inExamples 18-20 and 22 are shown in FIG. 11. In this figure, theconcentration-surface tension curve of the proteinaceous emulsifierobtained in Example 18 is denoted by A, that of the proteinaceousemulsifier obtained in Example 19 by B, that of the proteinaceousemulsifier obtained in Exaple 20 by C, that of gelatin by D, and that ofthe proteinaceous emulsifier obtained in Example 22 by E. By comparisonof the curves A, B, C, D and E, it can be seen that the proteinaceousemulsifier obtained in Examples 18, 19 and 20 are markedly superior insurface activity to the proteinaceous emulsifier obtained in Example 22and gelatin, and hence have excellent emulsifying power.

EXAMPLE 23 [PROTEINACEOUS EMULSIFIER 14]

A free L-alanine ester was prepared in manner described below and thenused in an enzymatic reaction.

(Synthesis of a Free L-Alanine Ester)

4.46 g (0.05 mole) of L-alanine was taken, 11.4 g (0.06 mole) ofp-toluenesulfonic acid hydrate was taken, and 11.79 g (0.055 mole) ofmyristyl alcohol was taken as an alcohol. After 200 ml of benzene wasadded thereto as a solvent, these materials were stirred and mixed well.The resulting reaction mixture was heated at the reflux temperature ofbenzene to carry out the esterification reaction. In this case, waterwas formed with the progress of the esterification reaction and releasedin the form of an azeotropic mixture with benzene, which wascontinuously removed from the reaction system. Thus, the reaction wascompleted in about 7-15 hours. After being cooled to room temperature,the reaction mixture was poured into a separating funnel and treatedwith 200 ml of a mixture of 0.2N NaOH and 3M NaCl solutions. Thistreatment was repeated three times. Thereafter, the benzene layer wasisolated and washed thoroughly with water. After washing with water, thebenzene layer was isolated and dried overnight over anhydrous sodiumsulfate. After drying, the benzene layer was filtered and the resultingbenzene solution was evaporated to remove the solvent. Thus, there wasobtained 13.7 g (96% yield) of purified free L-alanine myristyl ester inthe form of an oily material.

(Enzymatic Reaction)

120 ml of acetone was added to 480 ml of purified water and theresulting mixture was kept 37° C. with stirring. 120 g of gelatin as ahydrophilic protein and 34.26 g(i.e., 1 mole per 1,000 g of the gelatin)of L-alanine myristyl ester were added thereto and homogenized byadequate stirring. The resulting reaction mixture was adjusted to pH 9.0with 1M Na₂ CO₃.

After the addition of 1.05 g of L-cysteine hydochloride monohydrate and1.87 g of papain (manufactured by Nagase Sangyo K.K.), the aforesaidreaction mixture was stirred at 37° C. for 20 minutes. Then, thereaction was stopped by bringing the pH to 2.0 with 2N hydrochloricacid. The volume of the reaction mixture was about 1.5 liters.

While being kept at about 40° C., this reaction mixture was fed to ahollow-fiber ultrafiltration device (using a Cuprofan membrane andhaving an internal fiber diameter of 200μ, a membrane thickness of 11μand an effective membrane area of 1.5 m²) at a flow rate of about 10ml/min. As the outer fluid, warm water at about 40° C. was fed at a flowrate of about 1 liter/min. After completion of the dialysis, theinternal pressure of the same hollow-fiber ultrafiltration device wasadjusted to about 2 kg/cm² and the dialyzed solution was concentratedunder the same conditions as described above. Finally, about 300 ml of aconcentrated solution was obtained.

This concentrated solution was dried with a spray dryer to obtainProteinaceous Emulsifier 14.

EXAMPLE 24 [PROTEINACEOUS EMULSIFIERS 15, 16 AND 17]

Free L-leucine myristyl ester, L-glycine myristyl ester and L-isoleucinemyristyl ester were prepared in the same manner as in Example 23.

Then, using these esters, enzymatic reactions were carried out in thesame manner as in Example 23 to obtain Proteinaceous Emulsifiers 15, 16,and 17, respectively.

Emulsifying power was comparatively evaluated on ProteinaceousEmulsifier 14, 15 and 16 obtained in the above-described Examples 23 and24. These proteinaceous emulsifiers were composed of decompositionproducts of gelatin having a carboxyl end group joined to L-alaninemyristyl ester, L-leucine myristyl ester and L-glycine myristyl ester,respectively, by amide linkage.

(Procedure for Evaluation of Emulsifying Power)

1.8 parts by weight of liquid paraffin and a combined amount of 6.2 partby weight of a proteinaceous emulsifier and a 50 mM phosphate buffersolution (pH 7.0) were mixed so as to give a proteinaceous emulsifierconcentration of 1%, 3% or 5%. Using an ultradisperser, the resultingmixture was homogenized at 9,000 rpm at 80° C. for one minute and thencooled to room temperature. The sample so prepared was diluted 1:200with water and the absorbance of the diluted solution was measured witha spectrophotometer (Shimazu MPS-200).

The results thus obtained are shown in FIG. 12. As can be sent from thisfigure, the emulsifying power of Emulsifier 14 (L-alanine myristylester/gelatin), Emulsifier 15 (L-leucine myristyl ester/gelatin) andEmulsifier 16 (L-glycine myristyl ester/gelatin) decreases in thatorder.

EXAMPLE 25 [PROTEINACEOUS EMULSIFIER 18]

A free L-tyrosine ester was prepared in manner described below and thenused in an enzymatic reaction.

(Synthesis of a Free L-Tyrosine Ester)

9.1 g (0.05 mole) of L-tyrosine was used, 11.4 g (0.06 mole) ofp-toluenesulfonic acid hydrate was used, and 11.79 g (0.055 mole) ofmyristyl alcohol was used as an alcohol. After 200 ml of toluene wasadded thereto as a solvent, these materials were stirred and mixed well.The resulting reaction mixture was heated at the reflux temperature oftoluene to carry out the esterification reaction. In this case, waterwas formed with the progress of the esterification reaction and releasedin the form of an azeotropic mixture with toluene, which wascontinuously removed from the reaction system. Thus, the reaction wascompleted in about 7-15 hours. After being cooled to room temperature,the reaction mixture was poured into a separating funnel and treatedwith 200 ml of a mixture of 0.2N NaOH and 3M NaCl solutions. Thistreatment was repeated three times. Thereafter, the toluene layer wasisolated and washed thoroughly with water. After washing with water, thetoluene layer was isolated and dried overnight over anhydrous sodiumsulfate. After drying, the toluene layer was filtered and the resultingtoluene solution was evaporated to remove the solvent. Thus, there wasobtained 15.1 g (80% yield) of purified free L-tyrosine myristyl esterin the form of an oily material.

An infrared absorption spectrum of the free L-alanine cetyl ester thusobtained is shown in FIG. 13. In this figure, the peaks 21 and 22indicate the C═O stretching and C--O stretching vibrations originatingfrom the ester linkage.

The peaks 23, 24 and 25 indicate the NH stretching, NH deformation andC--N stretching vibrations, respectively, of a primary amine. The peak17 indicates the CH deformation vibration.

(Enzymatic Reaction)

120 ml of acetone was added to 480 ml of purified water and theresulting mixture was kept at 37° C. with stirring. 120 g of gelatin asa hydrophilic protein and 45.3 g (i.e., 1 mole per 1,000 g of thegelatin) of L-tyrosine myristyl ester were added thereto and homogenizedby adequate stirring. The resulting reaction mixture was adjusted to pH9.0 with 1M Na₂ CO₃.

After the addition of 1.05 g of L-cysteine hydrochloride monohydrate and1.57 g of papain (manufactured by Nagase Sangyo K.K.), the aforesaidreaction mixture was stirred at 37° C. for 20 minutes. Then, thereaction was stopped by bringing the pH to 2.0 with 2N HCl. The volumeof the reaction mixture was about 1.5 liters.

While being kept at about 40° C., this reaction mixture was fed to ahollow-fiber ultrafiltration device (using a Cuprofan membrane andhaving an internal fiber diameter of 200μ, a membrane thickness of 11μand an effective membrane area of 1.5 m²) at a flow rate of about 10ml/min. As the outer fluid, warm water at about 40° C. was fed at a flowrate of about 1 liter/min. After completion of the dialysis, theinternal pressure of the same hollow-fiber ultrafiltration device wasadjusted to about 2 kg/cm² and the dialyzed solution was concentratedunder the same conditions as described above. Finally, about 800 ml of aconcentrated solution was obtained.

This concentrated solution was dried with a spray dryer to obtainProteinaceous Emulsifier 18.

We claim:
 1. An emulsion type cosmetic composition comprising an oilphase and an aqueous phase, said composition containing, as theemulsifier, a proteinaceous emulsifier composed of (1) a decompositionproduct of a hydrophilic protein, said decomposition product having acarboxyl end group, said carboxyl end group of said decompositionproduct being joined to (2) the amino group of an amino acid ester by anamide linkage, said amino acid ester having an alcohol residuecontaining 14 to 22 carbon atoms.
 2. An emulsion type cosmeticcomposition as claimed in claim 1 wherein said amino acid ester is anamino acid ester represented by the general formula

    R.sub.3 CH(NH.sub.2)COOR.sub.2                             ( 2)

where R₃ is a methyl, isopropyl, n-butyl, isobutyl, sec-butyl,1-hydroxyethyl, benzyl or p-hydroxybenzyl group and R₂ is a saturatedaliphatic hydrocarbon radical or unsaturated aliphatic hydrocarbonradical having 14 to 22 carbon atoms.
 3. An emulsion type cosmeticcomposition as claimed in claim 1 wherein said amino acid ester is analiphatic alcohol ester of alanine represented by the general formula##STR3## where R₄ is a saturated aliphatic hydrocarbon radical or anunsaturated aliphatic hydrocarbon radical having 14 to 22 carbon atoms.4. An emulsion type cosmetic composition as claimed in claim 1 whereinsaid amino acid ester is an aliphatic alcohol ester of leucinerepresented by the general formula ##STR4## where R₅ is a saturatedaliphatic hydrocarbon radical or an unsaturated aliphatic hydrocarbonradical having 14 to 22 carbon atoms.
 5. An emulsion type cosmeticcomposition as claimed in claim 1 wherein said amino acid ester isobtained by reacting an L-amino acid with an aliphatic alcohol.
 6. Anemulsion type cosmetic composition as claimed in claim 1 wherein saidhydrophilic protein is casein, gelatin, sericin, soluble collagen orzein.
 7. A cosmetic composition as claimed in claim 6 in which saiddecomposition product is an enzymatic decomposition product of saidhydrophilic protein.
 8. An emulsion type cosmetic composition as claimedin claim 1, where the decomposition product is an enzymaticdecomposition product obtained by contacting said protein with anendopeptidase.
 9. An emulsion type cosmetic composition as claimed inclaim 8, where the endopeptidase is a thiol protease.
 10. An emulsiontype cosmetic composition as claimed in claim 9, where the thiolprotease is one or more selected from the group consisting of papain,ficin, cathepsin B, kiwi fruit protease, bromelain, chymopapain,cathepsin L, yeast proteinase B, cathepsin S and TZ-peptidase.
 11. Anemulsion type cosmetic composition as claimed in claim 9 where the thiolprotease is papain.
 12. An emulsion type cosmetic composition as claimedin claim 1 containing from 1 to 2% by weight of said emulsifier.
 13. Acosmetic composition as claimed in claim 1 wherein said amino acid esteris an amino acid ester represented by the general formula

    R.sub.1 CH(NH.sub.2)COOR.sub.2                             ( 1)

where R₁ is hydrogen, an alkyl group, an ω-hydroxyalkyl group, anaralkyl group or an ω-hydroxyaralkyl group and R₂ is a saturatedhydrocarbon or unsaturated hydrocarbon radical having 14 to 22 carbonatoms.